CD3 specific recombinant antibody

ABSTRACT

The invention provides recombinant antibody molecules comprising antigen binding regions derived from the heavy and/or light chain variable regions of a donor anti-CD3 antibody, e.g. OKT3, and which have anti-CD3 binding specificity, preferably of affinity similar to that of OKT3. The recombinant antibody is preferably a humanized antibody and may be a chimeric or CDR-grafted antibody. A method is disclosed for preparing CDR-grafted humanized antibodies in which, in addition to the CDR&#39;s non-human antibody residues are preferably used at positions 23, 24, 49, 71, 73 and 78 of the heavy chain variable region and at positions 46, 48, 58, and 71 of the light chain variable region. The recombinant, especially the humanized, anti-CD3 antibodies may be used for in vivo therapy or diagnosis.

This Application: is a continuation of U.S. provisional ApplicationSerial No. 08/116,247, Filed: Sep. 3, 1993, now U.S. Pat. No. 5,929,212,which is a File Wrapper Continuation of Ser. No. 07/743,377, Filed Oct.4, 1991, which claims priority from PCT/GB90/02018, Filed Dec. 21, 1990and GBRI Application No. 8928874.0, Filed Dec. 21, 1989.

FIELD OF THE INVENTION

The present invention relates to a recombinant antibody molecule (RAM),and especially a humanised antibody molecule (HAM), having specificityfor an antigen present in the T-cell receptor-CD3 complex of mostT-cells, to a process for its production using recombinant DNAtechnology and to its therapeutic use.

In the present application, the term “recombinant antibody molecule”(RAM) is used to describe an antibody produced by an process involvingthe use of recombinant DNA technology, including any analogues ofnatural immunoglobulins or their fragments. The term “humanised antibodymolecule” (HAM) is used to describe a molecule having an antigen bindingsite derived from an immunoglobulin from a non-human species, remainingimmunoglobulin-derived parts of the molecule being derived from a humanimmunoglobulin. The antigen binding site may comprise either completevariable domains fused onto constant domains or one or morecomplementarity determining regions grafted onto appropriate frameworkregions in the variable domains. The abbreviation “MAb” is used toindicate a monoclonal antibody.

In the description, reference is made to a number of publications bynumber. The publications are listed in numerical order at the end of thedescription.

BACKGROUND OF THE INVENTION

Natural immunoglobulins have been known for many years, as have thevarious fragments thereof, such as the Fab, (Fab′)₂ and Fc fragments,which can be derived by enzymatic cleavage. Natural immunoglobulinscomprise a generally Y-shaped molecule having an antigen-binding sitetowards the end of each upper arm. The remainder of the structure, andparticularly the stem of the Y, mediates the effector functionsassociated with immunoglobulins.

Natural immunoglobulins have been used in assay, diagnosis and, to amore limited extent, therapy. However, such uses, especially in therapy,have been hindered by the polyclonal nature of natural immunoglobulins.A significant step towards the realisation of the potential ofimmunoglobulins as therapeutic agents was the discovery of techniquesfor the preparation of monoclonal antibodies of defined specificity(ref. 1). However, most MAbs are produced by fusions of rodent spleencells with rodent myeloma cells. They are therefore essentially rodentproteins. There are very few reports of the production of human MAbs.

Since most available MAbs are of rodent origin, they are naturallyantigenic in humans and thus can give rise to an undesirable immuneresponse termed the HAMA (Human Anti-Mouse Antibody) response.Therefore, the use of rodent MAbs as therapeutic agents in humans isinherently limited by the fact that the human subject will mount animmunological response to the MAb and will either remove it entirely orat least reduce its effectiveness. Thus, in practice, MAbs of rodentorigin may not be used in patients for more than one or a few treatmentsas a HAMA response soon develops rendering the MAb ineffective as wellas giving rise to undesirable reactions.

Proposals have therefore been made for making non-human MAbs lessantigenic in humans. Such techniques can be generically termed“humanisation” techniques. These techniques generally involve the use ofrecombinant DNA technology to manipulate DNA sequences encoding thepolypeptide chains of the antibody molecule.

Early methods for humanising MAbs involved production of chimericantibodies in which an antigen binding site comprising the completevariable domains of one antibody is linked to constant domains derivedfrom another antibody. Methods for carrying out such chimerisationprocedures are described in EPO120694 (Celltech Limited), EP0125023(Genetech Inc. and City of Hope), EP-A-0 171496 (Res. Dev. Corp. Japan),EP-A-0 173 494 (Stanford University), and WO 86/01533 (CelltechLimited). This latter Celltech application (WO 86/01533) discloses aprocess for preparing an antibody molecule having the variable domainsfrom a mouse MAb and the constant domains from a human immunoglobulin.Such humanised chimeric antibodies, however, still contain a significantproportion of non-human amino acid sequence, i.e. the complete non-humanvariable domains, and thus may still elicit non-human variable domains,and thus may still elicit some HAMA response, particularly ifadministered over a prolonged period [Begent al al Br. J.Cancer, 62: 487(1990)].

WO 86/01533 also describes the production of an antibody moleculecomprising the variable domains of a mouse MAb, the CHl and CL domainsof a human immunoglobulin, and a non-immunoglobulin-derived protein inplace of the Fc portion of the human immunoglobulin.

In an alternative approach, described in EP-A-0239400 (Winter), thecomplementarity determining regions (CDRs) of a mouse MAb have beengrafted onto the framework regions of the variable domains of a humanimmunoglobulin by site directed mutagenesis using long oligonucleotides.There are 3 CDRs (CDR1, CDR2 and CDR3) in each of the heavy and lightchain variable regions. Such CDR-grafted humanised antibodies are muchless likely to give rise to a HAMA response than humanised chimericantibodies in view of the much lower proportion of non-human amino acidsequence which they contain.

The earliest work on humanising MAbs by CDR-grafting was carried out onMAbs recognising synthetic antigens, such as the NP or NIP antigens.However, examples in which a mouse MAb recognising lysozyme and a ratMAb recognising an antigen on human T-cells respectively were humanisedby CDR-grafting are shown by Verhoeyen et al (ref. 2) and Riechmann etal (ref. 3). The preparation of CDR-grafted antibody to the antigen onhuman T cells is also described in WO 89/07452 (Medical ResearchCouncil).

In Riechmann et al it was found that transfer of the CDR regions alone(as defined by Kabat refs. 4 and 5) was not sufficient to providesatisfactory antigen binding activity in the CDR-grafted product.Riechmann et al found that it was necessary to convert a serine residueat position 27 of the human sequence to the corresponding ratphenylalanine residue to obtain a CDR-grafted product havingsatisfactory antigen binding activity. This residue at position 27 ofthe heavy chain is within the structural loop adjacent to CDR1. Afurther construct which additionally contained a human serine to rattyrosine change at position 30 of the heavy chain did not have asignificantly altered binding activity over the humanised antibody withthe serine to phenylalanine change at position 27 alone. These resultsindicate that changes to residues of the human sequence outside the CDRregions, in particular in the loop adjacent to CFR1, may be necessary toobtain effective antigen binding activity for CDR-grafted antibodieswhich recognise more complex antigens. Even so the binding affinity ofthe best CDR-grafted antibodies obtained was still significantly lessthan the original MAb.

Very recently Queen et al (ref. 6) have described the preparation of ahumanised antibody that binds to the interleukin 2 receptor, bycombining the CDRs of a murine MAb (anti-Tac) with human immunoglobulinframework and constant regions. The human framework regions were chosento maximise homology with the anti-Tac MAb sequence. In additioncomputer modelling was used to identify framework amino acid residueswhich were likely to interact with the CDRs or antigen, and mouse aminoacids were used at these positions in the humanised antibody.

In WO 90/07861 Queen et al propose four criteria for designing humanisedimmunoglobulins. The first criterion is to use as the human acceptor theframework from a particular human immunoglobulin that is unusuallyhomologous to the non-human donor immunoglobulin to be humanised, or touse a consensus framework from many human antibodies. The secondcriterion is to use the donor amino acid rather than the acceptor if thehuman acceptor residue is unusual and the donor residue is typical forhuman sequences at a specific residue of the framework. The thirdcriterion is to use the donor framework amino acid residue rather thanthe acceptor at positions immediately adjacent to the CDRs. The fourthcriterion is to use the donor amino acid residue at framework positionsat which the amino acid is predicted to have a side chain atom withinabout 3 Å of the CDRs in a three-dimensional immunoglobulin model and tobe capable of interacting with the antigen or with the CDRs of thehumanised immunoglobulin. It is proposed that criteria two, three offour may be applied in addition or alternatively to criterion one, andmay be applied singly or in any combination.

WO 90/07861 described in detail the preparation of a single CDR-graftedhumanised antibody, a humanised antibody having specificity for the p55Tac protein of the IL-2 receptor. The combination of all four criteria,as above, were employed in designing this humanised antibody, thevariable region frameworks of the human antibody Eu (refs. 4 & 5) beingused as acceptor. In the resultant humanised antibody the donor CDRswere as defined by Kabat et al (refs. 4 and 5) and in addition the mousedonor residues were used in place of the human acceptor residues, atpositions 27, 30, 48, 66, 67, 89, 91, 94, 103, 104, 105 and 107 in theheavy chain and at positions 48, 60 and 63 in the light chain, of thevariable region frameworks. The humanised anti-Tac antibody obtained isreported to have an affinity for p55 of 3×10⁹ M⁻¹, about one-third ofthat of the murine MAb.

OKT3 is a mouse IgG2a/k MAb which recognises an antigen in the T-cellreceptor-CD3 complex and has been approved for use in many countriesthroughout the world as an immunosuppressant in the treatment of acuteallograft rejection [Chatenoud et al (ref. 7), and Jeffers et al (ref.8) However, in view of the murine nature of this MAb, a significant HAMAresponse, with a major anti-idiotype component, may build up on use.Clearly, it would be highly desirable to diminish or abolish thisundesirable HAMA response by suitable humanisation or other recombinantDNA manipulation of this very useful antibody and thus enlarge its areaof use. It would also be desirable to apply the techniques ofrecombinant DNA technology more generally to this useful antibody toprepare RAM products.

Moreover, we have further investigated the preparation of CDR-graftedhumanised antibody molecules and have identified a hierarchy ofpositions within the framework of the variable regions (i.e. outsideboth the Kabat CDRs and structural loops of the variable regions) atwhich the amino acid identities of the residues are important forobtaining CDR-grafted products with satisfactory binding affinity. Thishas enabled us to establish a protocol for obtaining satisfactoryCDR-grafted products which may be applied very widely irrespective ofthe level of homology between the donor immunoglobulin and acceptorframework. The set of residues which we have identified as being ofcritical importance does not coincide with the residues identified byQueen et al (ref. 6).

SUMMARY OF THE INVENTION

Accordingly the present invention provides an RAM comprising antigenbinding regions derived from the heavy and/or light chain variableregions of a donor anti-CD3 antibody and having anti-CD3 bindingspecificity, and preferably having an anti-CD3 binding affinity similarto that of OKT3.

Typically the donor anti-CD3 antibody is a rodent MAb.

The RAM of the invention may comprise antigen binding regions from anysuitable anti-CD3 antibody, typically a rodent anti-CD3 MAb, e.g. amouse or rat anti-CD3 MAb. The RAM may comprise a recombinant version ofwhole or a major part of the amino acid sequence of such a MAb. Also theRAM may comprise only the variable region (VH and/or VL) or one or moreCDRs of such a MAb. Especially the RAM may comprise amino acidsequences, whether variable region, CDR or other, derived from thespecific anti-CD3 MAb (OKT3) hereinafter specifically described withreference to FIGS. 1 and 2.

Preferably the RAM of the present invention is a humanised antibodymolecule (HAM) having specificity for CD3 and having an antigen bindingsite wherein at least one of the complementarity determining regions(CDRs) of the variable domain, usually at least two and preferably allof the CDRs, are derived from a non-human anti-CD3 antibody, e.g. arodent anti-CD3 MAb.

The RAM may be a chimeric antibody or a CDR-grafted antibody.

Accordingly, in preferred embodiments the invention provides an anti-CD3CDR-grafted antibody heavy chain having a variable region domaincomprising acceptor framework and donor CD3 binding regions wherein theframework comprises donor residues at at least one of positions 6, 23and/or 24, 48 and/or 49, 71 and/or 73, 75 and/or 76 and/or 78 and 88and/or 91.

More preferably, the heavy chain framework of the preferred embodimentcomprises donor residues at positions 23, 24, 49, 71, 73 and 78 or atpositions 23, 24 and 49. The residues at positions 71, 73 and 78 of theheavy chain framework are preferably either all acceptor or all donorresidues.

In particularly preferred embodiments the heavy chain frameworkadditionally comprises donor residues at one, some or all of positions6, 37, 48 and 94. Also it is particularly preferred that residues atpositions of the heavy chain framework which are commonly conservedacross species, i.e. positions 2, 4, 25, 36, 39, 47, 93, 103, 104, 106and 107, if not conserved between donor and acceptor, additionallycomprise donor residues. Most preferably the heavy chain frameworkadditionally comprises donor residues at positions 2, 4, 6, 25, 36, 37,39, 47, 48, 93, 94, 103, 104, 106 and 107.

In addition the heavy chain framework optionally comprises donorresidues at one, some or all of positions:

1 and 3,

72 and 76,

69 (if 48 is different between donor and acceptor),

38 and 46 (if 48 is the donor residue),

80 and 20 (if 69 is the donor residue),

67,

82 and 18 (if 67 is the donor residue),

91,

88, and

any one or more of 9, 11, 41, 87, 108, 110 and 112.

In the preferred embodiments of the present invention described aboveand hereinafter, reference is made to CDR-grafted antibody productscomprising acceptor framework and donor antigen binding regions. It willbe appreciated that the invention is widely applicable to theCDR-grafting of anti-CD3 antibodies in general. Thus, the donor andacceptor antibodies may be anti-CD3 antibodies derived from animals ofthe same species and even same antibody class or sub-class. Moreusually, however, the donor and acceptor antibodies are derived fromanimals of different species. Typically, the donor anti-CD3 antibody isa non-human antibody, such as a rodent MAb, and the acceptor antibody isa human antibody.

In the CDR-grafted antibody products of the present invention, the donorCD3 binding region typically comprises at least one CDR from the donorantibody. Usually the donor antigen binding region comprises at leasttwo and preferably all three CDRs of each of the heavy chain and/orlight chain variable regions. The CDRs may comprise the Kabat CDRs, thestructural loop CDRs or a composite of the Kabat and structural loopCDRs and any combination of any of these. Preferably, the antigenbinding regions of the CDR-grafted heavy chain variable domain compriseCDRs corresponding to the Kabat CDRs at CDR2 (residues 50-65) and CDR3(residues 95-100) and a composite of the Kabat and structural loop CDRsat CDR1 (residues 26-35).

The residue designations given above and elsewhere in the presentapplication are numbered according to the Kabat numbering (refs. 4 and5). Thus the residue designations do not always correspond directly withthe linear numbering of the amino acid residues. The actual linear aminoacid sequence may contain fewer or additional amino acids than in thestrict Kabat numbering corresponding to a shortening of, or insertioninto, a structural component, whether framework or CDR, of the basicvariable domain structure. For example, the heavy chain variable regionof the anti-Tac antibody described by Queen et al (ref. 6) contains asingle amino acid insert (residue 52a) after reside 52 of CDR2 and athree amino acid insert (residues 82a, 82b and 82c) after frameworkresidue 82, in the Kabat numbering. The correct Kabat numbering ofresidues may be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The invention also provides in a further preferred embodiment aCDR-grafted antibody light chain having a variable region domaincomprising acceptor framework and donor CD3 binding regions wherein theframework comprises donor residues at at least one of positions 1 and/or3 and 46 and/or 47. Preferably the CDR grafted light chain of thispreferred embodiment comprises donor residues at positions 46 and/or 47.

The invention also provides in a yet further preferred embodiment aCDR-grafted antibody light chain having a variable region domaincomprising acceptor framework and donor CD3 binding regions wherein theframework comprises donor residues at at least one of positions 46, 48,58 and 71.

More preferably in this latter embodiment, the framework comprises donorresidues at all of positions 46, 48, 58 and 71.

In particularly preferred embodiments of the above preferredembodiments, the light chain framework additionally comprises donorresidues at positions 36, 44, 47, 85 and 87. Similarly positions of thelight chain framework which are commonly conserved across species, i.e.positions 2, 4, 6, 35, 49, 62, 64-69, 98, 99, 101 and 102, if notconserved between donor and acceptor, additionally comprise donorresidues. Most preferably the light chain framework additionallycomprises donor residues at positions 2, 4, 6, 35, 36, 38, 44, 47, 49,62, 64-69, 85, 87, 98, 99, 101 and 102.

In additions the light chain framework of the above preferredembodiments optionally comprises donor residues at one, some or all ofpositions:

1 and 3,

63,

60 (if 60 and 54 are able to form at potential saltbridge),

70 (if 70 and 24 are able to form a potential saltbridge),

73 and 21 (if 47 is different between donor and acceptor),

37 and 45 (if 47 is different between donor and acceptor), and

any one or more of 10, 12, 40, 80, 103 and 105.

Preferably, the antigen binding regions of the CDR-grafted light chainvariable domain comprise CDRs corresponding to the Kabat CDRs at CDR1(residue 24-34), CDR2 (residues 50-56) and CDR3 (residues 89-97).

The invention further provides in a fourth aspect a CDR-grafted antibodymolecule comprising at least one CDR-grafted heavy chain and at leastone CDR-grafted light chain as defined above.

The CDR-grafted and humanised antibody molecules and chains of thepresent invention may comprise: a complete antibody molecule, havingfull length heavy and light chains; a fragment thereof, such as a Fab,(Fab′)₂ or FV fragment; a light chain or heavy chain monomer or dimer;or a single chain antibody, e.g. a single chain FV in which heavy andlight chain variable regions are joined by a peptide linker; or anyother CDR-grafted or humanised antibody product with anti-CD3 bindingspecificity. Similarly the CDR-grafted heavy and light chain variableregion may be combined with other antibody domains as appropriate.

Also the CDR-grafted or humanised heavy or light chains or antibodymolecules of the present invention may have attached to them an effectoror reporter molecule. For instance, it may have a macrocycle, forchelating a heavy metal atom, or a toxin, such as ricin, attached to itby a covalent bridging structure. Alternatively, the procedures ofrecombinant DNA technology may be used to produce an immunoglobulinmolecule in which the Fc fragment of CH3 domain of a completeimmunoglobulin molecule has been replaced by, or has attached thereto bypeptide linkage, a functional non-immunoglobulin protein, such as anenzyme or toxin molecule.

For CDR-grafted antibody products, any appropriate acceptor variableregion framework sequences may be used having regard to class/type ofthe donor antibody from which the antigen binding regions are derived.Preferably, the type of acceptor framework used is of the same/similarclass/type as the donor antibody. Conveniently, the framework may bechosen to maximise/optimise homology with the donor antibody sequenceparticularly at positions close or adjacent to the CDRs. However, a highlevel of homology between donor and acceptor sequences is not importantfor application of the present invention. The present inventionidentifies a hierarchy of framework residue positions at which donorresidues may be important or desirable for obtaining a CDR-graftedantibody product having satisfactory binding properties. The presentinvention advantageously enables the preparation of CDR-grafted antibodyproducts having binding affinities similar to, and even in some casesbetter than the corresponding donor antibody product, e.g. OKT3 product.Preferably, the CDR-grafted antibody products of the invention havebinding affinities of at least about 10⁵ M⁻¹, preferably at least about10⁸ M⁻¹ and especially within the range 10⁸−10¹² M⁻¹. In principle, thepresent invention is applicable to any combination of donor and acceptorantibodies irrespective of the level of homology between theirsequences. A protocol for applying the invention to any particulardonor-acceptor antibody pair is given hereinafter. Examples of humanframeworks which may be used are KOL, NEWM, REI, EU, LAY and POM (refs.4 and 5); for instance KOL and NEWM for the heavy chain and RE1 for thelight chain and EU, LAY and POM for both the heavy chain and the lightchain.

Also the constant region domains of the products of the invention may beselected having regard to the proposed function of the antibody inparticular the effector functions which may be required. For example,the constant region domains may be human IgA, IgE, IgG or IgM domains.In particular, IgG human constant region domains may be used, especiallyof the IgG1 and IgG3 isotypes, when the humanised antibody molecule isintended for therapeutic uses, and antibody effector functions arerequired. Alternatively, IgG2 and IgG4 isotypes may be used when thehumanised antibody molecule is intended for therapeutic purposes andantibody effector functions are not required, e.g. for simple blockingof the T-cell receptor-CD3 complex.

However, the remainder of the antibody molecules need not comprise onlyprotein sequences from immunoglobulins. For instance, a gene may beconstructed in which a DNA sequence encoding part of a humanimmunoglobulin chain is fused to a DNA sequence encoding the amino acidsequence of a polypeptide effector or reporter molecule.

Preferably the CDR-grafted heavy and light chain and antibody moleculeproducts are produced by recombinant DNA technology.

Thus in further aspects the invention also includes DNA sequences codingfor the RAMs, HAMs and CDR-grafted heavy and light chains, cloning andexpression vectors containing the DNA sequences, host cells transformedwith the DNA sequences and processes for producing the CDR-graftedantibody molecules comprising expressing the DNA sequences in thetransformed host cells.

The general methods by which the vectors may be constructed,transfection methods and culture methods are well known per se and formno part of the invention. Such methods are shown, for instance, inreferences 9 and 10.

The DNA sequences which encode the anti-CD3 donor amino acid sequencesmay be obtained by methods well known in the art. For example theanti-CD3 coding sequences may be obtained by genomic cloning, or cDNAcloning from suitable hybridoma cell lines, e.g. the OKT3 cell linehereinafter specifically described. Positive clones may be screenedusing appropriate probes for the heavy and light chain genes inquestion. Also PCR cloning may be used.

DNA coding for acceptor, e.g. human acceptor, sequences may be obtainedin any appropriate way. For example DNA sequences coding for preferredhuman acceptor frameworks such as KOL, REI, EU and NEWM, are widelyavailable to workers in the art.

The standard techniques of molecular biology may be used to prepare DNAsequences coding for the chimeric and CDR-grafted products. Desired DNAsequences may be synthesised completely or in part using oligonucleotidesynthesis techniques. Site-directed mutagenesis and polymerase chainreaction (PCR) techniques may be used as appropriate. For exampleoligonucleotide directed synthesis as described by Jones et al (ref. 17)may be used. Also oligonucleotide directed mutagenesis of a pre-exisingvariable region as, for example, described by Verhoeyen et al (ref. 2)or Riechmann et al (ref. 3) may be used. Also enzymatic filling in ofgapped oligonucleotides using T4 DNA polymerase as, for example,described by Queen et al (ref. 6) may be used.

Any suitable host cell/vector system may be used for expression of theDNA sequences coding for the CDR-grafted heavy and light chains.Bacterial e.g. E. coli, and other microbial systems may be used, inparticular for expression of antibody fragments such as FAb and (Fab′)₂fragments, and especially FV fragments and single chain antibodyfragments e.g. single chain FVs. Eucaryotic, e.g. mammalian, host cellexpression systems may be used, in particular, for production of largerCDR-grafted antibody products, including complete antibody molecules.Suitable mammalian host cells include CHO cells and myeloma or hybridomacell lines.

Thus, according to a further aspect the present invention provides aprocess for producing an anti-CD3 RAM which process comprises:

(a) producing in an expression vector an operon having a DNA sequencewhich encodes an antibody heavy chain wherein at least one CDR of thevariable domain is derived from a donor anti-CD3 antibody and remainingimmunglobulin-derived parts of the antibody chain are derived from anacceptor immunoglobulin;

and/or

(b) producing in an expression vector an operon having a DNA sequencewhich encodes a complementary antibody light chain wherein at least oneCDR of the variable domain is derived from a donor anti-CD3 antibody andthe remaining immunoglobulin-derived parts of the antibody chain arederived from an acceptor immunoglobulin;

(c) transfecting a host cell with the or each vector;

and

(d) culturing the transfected cell line to produce the RAM.

The RAM may comprise only heavy or light chain-derived polypeptide, inwhich case only a heavy chain or light chain polypeptide coding sequenceis used to transfect the host cells.

For production of RAMs comprising both heavy and light chains, the cellline may be transfected with two vectors. The first vector may containan operon encoding a light chain-derived polypeptide and the secondvector may contain an operon encoding a heavy chain-derived polypeptide.Preferably, the vectors are identical except in so far as the codingsequences and selectable markers are concerned so as to ensure as far aspossible that each polypeptide chain is equally expressed.Alternatively, a single vector may be used, the vector including thesequences encoding both light chain- and heavy chain-derivedpolypeptides.

The DNA in the coding sequences for the light and heavy chains maycomprise cDNA or genomic DNA or both. However, it is preferred that theDNA sequence encoding the heavy or light chain comprises, at leastpartially, genomic DNA. Most preferably, the heavy or light chainencoding sequence comprises a fusion of cDNA and genomic DNA.

The present invention also includes therapeutic and diagnosticcompositions comprising the RAMs, HAMs and CDR-grafted light and heavychains and molecules of the invention and uses of such compositions intherapy and diagnosis.

Accordingly in a further aspect the invention provides a therapeutic ordiagnostic composition comprising a RAM, HAM or CDR-grafted antibodyheavy or light chain or molecule according to previous aspects of theinvention in combination with a pharmaceutically acceptable carrier,diluent or excipient.

Accordingly also the invention provides a method of therapy or diagnosiscomprising administering an effective amount of a RAM, HAM orCDR-grafted antibody heavy or light chain or molecule according toprevious aspects of the invention to a human or animal subject.

The RAM, HAM and CDR-grafted products of the present invention may beused for any of the therapeutic uses for which anti CD3 antibodies, e.g.OKT3, have been used or may be used in the future. For example, theproducts may be used as ummunosuppressants, e.g. in the treatment ofacute allograft rejection.

A preferred protocol for obtaining CDR-grafted antibody heavy and lightchains in accordance with the present invention is set out belowtogether with the rationale by which we have derived this protocol. Thisprotocol and rationale are given without prejudice to the generality ofthe invention as hereinbefore described and defined.

Protocol

It is first of all necessary to sequence the DNA coding for the heavyand light chain variable regions of the donor antibody, to determinetheir amino acid sequences. It is also necessary to choose appropriateacceptor heavy and light chain variable regions, of known amino acidsequence. The CDR-grafted chain is then designed starting from the basisof the acceptor sequence. It will be appreciated that in some cases thedonor and acceptor amino acid residues may be identical at a particularposition and thus no change of acceptor framework residue is required.

1. As a first step donor residues are substituted for acceptor residuesin the CDRs. For this purpose the CDRs are preferably defined asfollows:

Heavy chain CDR1: residues 26-35 CDR2: residues 50-65 CDR3: residues95-102 Light chain CDR1: residues 24-34 CDR2: residues 50-56 CDR3:residues 89-97

The positions at which donor residues are to be substituted for acceptorin the framework are then chosen as follows, first of all with respectto the heavy chain and subsequently with respect to the light chain.

2. Heavy Chain

2.1 Choose donor residues at all of positions 23, 24, 49, 71, 73 and 78of the heavy chain or all of positions 23, 24 and 49 (71, 73 and 78 areeither all donor or all acceptor).

2.2 Check that the following have the same amino acid in donor andacceptor sequences, and if not preferably choose the donor: 2, 4, 6, 25,36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.

2.3 To further optimise affinity consider choosing donor residues atone, some or any of:

i. 1, 3

ii. 72, 76

iii. If 48 is different between donor and acceptor sequences, consider69

iv. If at 48 the donor residue is chosen, consider 38 and 46

v. If at 69 the donor residue is chosen, consider 80 and then 20

vi. 67

vii. If at 67 the donor residue is chosen, consider 82 and then 18

viii. 91

ix. 88

x. 9, 11, 41, 87, 108, 110, 112

3. Light Chain

3.1 Choose donor at 46, 48, 58 and 71

3.2 Check that the following have the same amino acid in donor andacceptor sequences, if not preferably choose donor: 2, 4, 6, 35, 38, 44,47, 49, 62, 64-69 inclusive, 85, 87, 98, 99, 101 and 102

3.3 To further optimise affinity consider choosing donor residues atone, some or any of:

i. 1, 3

ii. 63

iii. 60, if 60 and 54 are able to form a potential saltbridge

iv. 70, if 70 and 24 are able to form a potential saltbridge

v. 73 and 21, if 47 is different between donor and acceptor

vi. 37 and 45, if 47 is different between donor and acceptor

vii. 10, 12, 40, 80, 103, 105

Rationale

In order to transfer the binding site of an antibody into a differentacceptor framework, a number of factors need to be considered.

1. The extent of the CDRs

The CDRs (Complementary Determining Regions) were defined by Wu andKabat (refs. 4 and 5) on the basis of an analysis of the variability ofdifferent regions of antibody variable regions. Three regions per domainwere recognised. In the light chain the sequences are 24-34, 50-56,89-97 (numbering according to Kabat (ref. 4), Eu Index) inclusive and inthe heavy chain the sequences are 31-35, 50-65 and 95-102 inclusive.

When antibody structures became available it became apparent that theseCDR regions corresponded in the main to loop regions which extended fromthe β barrel framework of the light and heavy variable domains. For H1there was a discrepancy in that the loop was from 26 to 32 inclusive andfor H2 to loop was 52-56 and for L2 from 50 to 53. However, with theexception of H1 the CDR regions encompassed the loop regions andextended into the β strand frameworks. In H1 residue 26 tends to be aserine and 27 a phenylalanine or tyrosine, residue 29 is a phenylalaninein most cases. Residues 28 and 30 which are surface residues exposed tosolvent might be involved in antigen-binding. A prudent definition ofthe H1 CDR therefore would include residues 26-35 to include both theloop region and the hypervariable residues 33-35.

It is of interest to note the example of Riechmann et al (ref. 3), whoused the residue 31-35 choice for CDR-H1. In order to produce efficientantigen binding residue 27 also needed to be recruited from the donor(rat) antibody.

2. Non-CDR residues which contribute to antigen binding

By examination of available X-ray structures we have identified a numberof residues which may have an effect on net antigen binding and whichcan be demonstrated by experiment. These residues can be sub-dividedinto a number of groups.

2.1 Surface residues near CDR [all numbering as in Kabat et al (ref.4)].

2.1.1. Heavy Chain—Key residues are 23, 71 and 73. Other residues whichmay contribute to a lesser extent are 1, 3 and 76. Finally 25 is usuallyconserved but the murine residue should be used if there is adifference.

2.1.2 Light Chain—Many residues close to the CDRs, e.g. 63, 65, 67 and69 are conserved. If conserved none of the surface residues in the lightchain are likely to have a major effect. However, if the murine residueat these positions is unusual, then it would be of benefit to analysethe likely contribution more closely. Other residues which may alsocontribute to binding are 1 and 3, and also 60 and 70 if the residues atthese positions and at 54 and 24 respectively are potentially able toform a salt bridge i.e. 60+54; 70+24.

2.2 Packing residues near the CDRs.

2.2.1. Heavy Chain—Key residues are 24, 49 and 78. Other key residueswould be 36 if not a tryptophan, 94 if not an arginine, 104 and 106 ifnot glycines and 107 if not a threonine. Residues which may make afurther contribution to stable packing of the heavy chain and henceimproved affinity are 2, 4, 6, 38, 46, 67 and 69. 67 packs against theCDR residue 63 and this pair could be either both mouse or both human.Finally, residues which contribute to packing in this region but from alonger range are 18, 20, 80, 82 and 86. 82 packs against 67 and in turn18 packs against 82. 80 packs against 69 and in turn 20 packs against80. 86 forms an H bond network with 38 and 46. Many of the mouse-humandifferences appear minor e.g. Leu-Ile, but could have an minor impact oncorrect pacing which could translate into altered positioning of theCDRs.

2.2.2 Light Chain—Key residues are 48, 58 and 71. Other key residueswould be 6 if not glutamine, 35 if not tryptophan, 62 if notphenylalanine or tryosine, 64, 66, 68, 99 and 101 if not glycines and102 if not a threonine. Residues which make a further contribution are2, 4, 37, 45 and 47. Finally residues 73 and 21 and 19 may make longdistance packing contributions of a minor nature.

2.3. Residues at the variable domain interface between heavy and lightchains—In both the light and heavy chains most of the non-CDR interfaceresidues are conserved. If a conserved residue is replaced by a residueof different character, e.g. size or charge, it should be considered forretention as the murine residue.

2.3.1 Heavy Chain—Residues which need to be considered are 37 if theresidue is not a valine but is of larger side chain volume or has acharge or polarity. Other residues are 39 if not a glutamine, 45 if nota leucine, 47 if not a tryptophan, 91 if not a phenylalanine ortyrosine, 93 if not an alanine and 103 if not a tryptophan. Residue 89is also at the interface but is not in a position where the side chaincould be of great impact.

2.3.2. Light Chain—Residues which need to be considered are 36, if not atyrosine, 38 if not a glutamine, 44 if not a proline, 46, 49 if not atyrosine, residue 85, residue 87 if not a tyrosine and 98 if not aphenylalanine.

2.4. Variable-Constant region interface—The elbow angle between variableand constant regions may be affected by alterations in packing of keyresidues in the variable region against the constant region which mayeffect the position of V_(L) and V_(H) with respect to one another.Therefore it is worth noting the residues likely to be in contact withthe constant region. In the heavy chain the surface residues potentiallyin contact with the variable region are conserved between mouse andhuman antibodies therefore the variable region contact residues mayinfluence the V-C interaction. In the light chain the amino acids foundat a number of the constant region contact points vary, and the V & Cregions are not in such close proximity as the heavy chain. Thereforethe influences of the light chain V-C interface may be minor.

2.4.1. Heavy Chain—Contact residues are 7, 11, 41, 87, 108, 110, 112.

2.4.2. Light Chain—In the light chain potentially contacting residuesare 10, 12, 40, 80, 83, 103 and 105.

The above analysis coupled with our considerable practical experimentalexperience in the CDR-grafting of a number of different antibodies havelead us to the protocol given above.

The present invention is now described, by way of example only, withreference to the accompanying FIGS. 1-13.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a and b shown in DNA and amino acid sequences of the OKT3 lightchain (SEQ ID NO:4 and 5):

FIGS. 2a and 6 show DNA and amino acid sequences of the OKT3 heavy chainSEQ ID NO:6 and 7);

FIG. 3 shows the alignment of the OKT3 light variable region amino acidsequence with that of the light variable region of the human antibodyREI;

FIG. 4 shows the alignment of the OKT3 heavy variable region amino acidsequence with that of the heavy variable region of the human antibodyKOL;

FIGS. 5A-C show the heavy variable region amino acid sequence of OKT3,KOL and various CDR grafts — SEQ ID NOs:10, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 11, respectively

FIG. 6 shows the light variable region amino acid sequences of OKT3, REIand various corresponding CDR grafts SEQ ID Nos:8, 26, 27, 28, 29, and9, respectively;

FIG. 7 shows a graph of binding assay results for various grafted OKT3antibodies′

FIG. 8 shows a graph of blocking assay results for various grafted OKT3antibodies;

FIG. 9 shows a similar graph of blocking assay results;

FIGS. 10 shows similar graphs for both binding assay and blocking assayresults;

FIGS. 11 shows further similar graphs for both binding assay andblocking assay results;

FIG. 12 shows a graph of competition assay results for a minimallygrafted OKT3 antibody compared with the OKT3 murine reference standard,and

FIG. 13 shows a similar graph of competition assay results comparing afully grafted OKT3 antibody with the murine reference standard.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION EXAMPLE Materialand Methods

1. INCOMING CELLS

Hybridoma cells producing antibody OKT3 were provided by Ortho (seedlot4882.1) and were grown up in antibiotic free Dulbecco's Modified EaglesMedium (DMEM) supplemented with glutamine and 5% foetal calf serum, anddivided to provide both an overgrown supernatant for evaluation andcells for extraction of RNA. The overgrown supernatant was shown tocontain 250 ug/mL murine IgG2a/kappa antibody. The supernatant wasnegative for murine lambda light chain and IgG1, IgG2b, IgG3, IgA andIgM heavy chain. 20 mL of supernatant was assayed to confirm that theantibody present was OKT3.

2. MOLECULAR BIOLOGY PROCEDURES

Basic molecular biology procedures were as described in Maniatis et al(ref. 9) with, in some cases, minor modifications. DNA sequencing wasperformed as described in Sanger et al (ref. 11) and the AmershamInternational Plc sequencing handbook. Site directed mutagenesis was asdescribed in Kramer et al (ref. 12) and the Anglian Biotechnology Ltd.handbook. COS cell expression and metabolic labelling studies were asdescribed in Whittle et al (ref. 13)

3. RESEARCH ASSAYS

3.1. ASSEMBLY ASSAYS

Assembly assays were performed on supernatants from transfected COScells to determine the amount of intact IgG present.

3.1.1. COST CELLS TRANSFECTED WITH MOUSE OKT3 GENES

The assembly assay for intact mouse IgG in COS cell supernatants was anELISA with the following format:

96 well microtitre plates were coated with F(ab′)₂ goat anti-mouse IgGFc. The plates were washed in water and samples added for 1 hour at roomtemperature. The plates were washed and F(ab′)₂ goat anti-mouse IgGF(ab′)₂ (HRPO conjugated) was then added. Substrate was added to revealthe reaction. UPC10, a mouse IgG2a myeloma, was used as a standard.

3.1.2. COS AND CHO CELLS TRANSFECTED WITH CHIMERIC OR CDR-GRAFTED OKT3GENES

The assembly assay for chimeric or CDR-grafted antibody in COS cellsupernatants was an ELISA with the following format:

96 well microtitre plates were coated with F(ab′)₂ goat anti-human IgGFc. The plates were washed and samples added and incubated for 1 hour atroom temperature. The plates were washed and monoclonal mouse anti-humankappa chain was added for 1 hour at room temperature. The plates werewashed and F(ab′)₂ goat anti-mouse IgG Fc (HRPO conjugated) was added.Enzyme substrate was added to reveal the reaction. Chimeric B72.3 (IgG4)(ref. 13) was used as a standard. The use of a monoclonal anti-kappachain in this assay allows grafted antibodies to be read from thechimeric standard.

3.2. ASSAY FOR ANTIGEN BINDING ACTIVITY

Material from COS cell supernatants was assayed for OKT3 antigen bindingactivity onto CD3 positive cells in a direct assay. The procedure was asfollows:

HUT 78 cells (human T cell line, CD3 positive) were maintained inculture. Monolayers of HUT 78 cells were prepared onto 96 well ELISAplates using poly-L-lysine and glutaraldehyde. Samples were added to themonolayers for 1 hour at room temperature. The plates were washed gentlyusing PBS. F(ab′)₂ goat anti-human IgG Fc (HRPO conjugated) or F(ab′)₂goat anti-mouse IgG Fc (HRPO conjugated) was added as appropriate forhumanised or mouse samples. Substrate was added to reveal the reaction.The negative control for the cell-based assay was chimer B72.3. Thepositive control was mouse Orthomune OKT3 or chimeric OKT3, whenavailable. This cell-based assay was difficult to perform, and analternative assay was developed for CDR-grafted OKT3 which was moresensitive and easier to carry out. In this system, CDR-grafted OKT3produced by COS cells was tested for its ability to bind to theCD3-positive HPB-ALL (human peripheral blood acute lymphocytic leukemia)cell line. It was also tested for its ability to block the binding ofmurine OKT3 to these cells. Binding was measured by the followingprocedure: HPB-ALL cells were harvested from tissue culture. Cells wereincubated at 4° C. for 1 hour with various dilutions of test antibody,positive control antibody, or negative control antibody. The cells werewashed once and incubated at 4° C. for 1 hour with an FITC-labelled goatanti-human IgG (Fc− specific, mouse absorbed). The cells were washedtwice and analysed by cytofluorography. Chimeric OKT3 was used as apositive control for direct binding. Cells incubated withmock-transfected COS cell supernatant, followed by the FITC-labelledgoat anti-human IgG, provided the negative control. To test the abilityof CDR-grafted OKT3 to block murine OKT3 binding, the HPB-ALL cells wereincubated at 4° C. for 1 hour with various dilutions of test antibody orcontrol antibody. A fixed saturating amount of FITC OKT3 was added. Thesamples were incubated for 1 hour at 4° C., washed twice and analysed bycytofluorography. FITC-labelled OKT3 was used as a positive control todetermine maximum binding. Unlabelled murine OKT3 served as a referencestandard for blocking. Negative controls were unstained cells with orwithout mock-transfected cell supernatant. The ability of theCDR-grafted OKT3 light chain to bind CD3-positive cells and block thebinding of murine OKT3 was initially tested in combination with thechimeric OKT3 heavy chain. The chimeric OKT3heavy chain is composed ofthe murine OKT3 variable region and the human IgG4 constant region. Thechimeric heavy chain gene is expressed in the same expression vectorused for the CDR-grafted genes. The CDR-grafted light chain expressionvector and the chimeric heavy chain expression vector wereco-transfected into COS cells. The fully chimeric OKT3 antibody(chimeric light chain and chimeric heavy chain) was found to be fullycapable of binding to CD3 positive cells and blocking the binding ofmurine OKT3 to these cells.

3.3 DETERMINATION OF RELATIVE BINDING AFFINITY

The relative binding affinities of CDR-grafted anti-CD3 monoclonalantibodies were determined by competition binding (ref. 6) using theEPB-ALL human T cell line as a source of CD3 antigen, andfluorescein-conjugated murine OKT3 (Fl-OKT3) of known binding affinityas a tracer antibody. The binding affinity of Fl-OKT3 tracer antibodywas determined by a direct binding assay in which increasing amounts ofFl-OKT3 were incubated with HPB-ALL (5×10⁵) in PBS with 5% foetal calfserum for 60 min. at 4° C. Cells were washed, and the fluorescenceintensity was determined on a FACScan flow cytometer calibrated withquantitative microbead standards (Flow Cytometry Standards, ResearchTriangle Park, N.C.). Fluorescence intensity per antibody molecule (F/Pratio) was determined by using microbeads which have a predeterminednumber of mouse IgG antibody binding sites (Simply Cellular beads, FlowCytometry Standards). F/P equals the fluorescence intensity of beadssaturated with Fl-OKT3 divided by the number of binding sites per bead.The amount of bound and free Fl-OKT3 was calculated from the meanfluorescence intensity per cell, and the ratio of bound/free was plottedagainst the number of moles of antibody bound. A linear fit was used todetermine the affinity of binding (absolute value of the slope).

For competitive binding, increasing amounts of competitor antibody wereadded to a sub-saturating dose of Fl-OKT3 and incubated with 5×10⁵HPB-ALL in 200 μl of PBS with 5% foetal calf serum, for 60 min at 4° C.The fluorescence intensities of the cells were measured on a FACScanflow cytometer calibrated with quantitative microbead standards.

The concentrations of bound and free Fl-OKT3 were calculated. Theaffinities of competing antibodies were calculated from the equation[X]−[OKT3]=(1/Kx)−(1/Ka), where Ka is the affinity of murine OKT3, Kx isthe affinity of competitor X, [] is the concentration of competitorantibody at which bound/free binding is R/2, and R is the maximalbound/free binding.

4. cDNA LIBRARY CONSTRUCTION

4.1. mRNA PREPARATION AND cDNA SYNTHESIS

OKT3 producing cells were grown as described above and 1.2×10⁹ cellsharvested and mRNA extracted using the guanidinium/LiCl extractionprocedure. cDNA was prepared by priming from Oligo-dT to generate fulllength cDNA. The cDNA was methylated and EcoR1 linkers added forcloning.

4.2. LIBRARY CONSTRUCTION

The cDNA library was ligated to pSP65 vector DNA which had been EcoR1cut and the 5′ phosphate groups removed by calf intestinal phosphatase(EcoR1/CIP). The ligation was used to transform high transformationefficiency Escherichia coli (E. coli) HB101. A cDNA library wasprepared. 3600 colonies were screened for the light chain and 10000colonies were screened for the heavy chain.

5. SCREENING

E. coli colonies positive for either heavy or light chain probes wereidentified by oligonucleotide screening using the oligonucleotides:

5′ TCCAGATGTTAACTGCTCAC (SEQ ID NO: 1) for the light chain, which iscomplementary to a sequence in the mouse kappa constant region, and 5′CAGGGGCCAGTGGATGGATAGAC (SEQ ID NO: 2) for the heavy chain which iscomplementary to a sequence in the mouse IgG2a constant CH1 domainregion. 12 light chain and 9 heavy chain clones were identified andtaken for second round screening. Positive clones from the second roundof screening were grown up and DNA prepared. The sizes of the geneinserts were estimated by gel electrophoresis and inserts of a sizecapable of containing a full length cDNA were subcloned into M13 for DNAsequencing.

6. DNA SEQUENCING

Clones representing four size classes for both heavy and light chainswere obtained in M13. DNA sequence for the 5′ untranslated regions,signal sequences, variable regions and 3′ untranslated regions of fulllength cDNAs [FIGS. 1(a) and 2(a)] were obtained and the correspondingamino acid sequences predicted [(FIGS. 1(b) and 2(b)]. In FIG. 1(a) theuntranslated DNA regions are shown in uppercase, and in both FIGS. 1 and2 the signal sequences are underlined.

7. CONSTRUCTION OF cDNA EXPRESSION VECTORS

Celltech expression vectors are based on the plasmid pEE6hCMV (ref. 14).A polylinker for the insertion of genes to be expressed has beenintroduced after the major immediate early promoter/enhancer of thehuman Cytomegalovirus (hCMV). Marker genes for selection of the plasmidin transfected eukaryotic cells can be inserted as BamH1 cassettes inthe unique BamH1 site of pEE6 hCMV; for instance, the neo marker toprovide pEE6 hCMV neo. It is usual practice to insert the neo and gptmarkers prior to insertion of the gene of interest, whereas the GSmarker is inserted last because of the presence of internal EcoR1 sitesin the cassette. The selectable markers are expressed from the SV40 latepromoter which also provides an origin of replication so that thevectors can be used for expression in the COS cell transient expressionsystem.

The mouse sequences were excised from the M13 based vectors describedabove as EcoR1 fragments and cloned into either pEE6-hCMV-neo for theheavy chain and into EE6-hCMV-gpt for the light chain to yield vectorspJA136 and pJA135 respectively.

8. EXPRESSION OF cDNAS IN COS CELLS

Plasmids pJA135 and pJA136 were co-transfected into COS cells andsupernatant from the transient expression experiment was shown tocontain assembled antibody which bound to T-cell enriched lymphocytes.Metabolic labelling experiments using ³⁵S methionine showed expressionand assembly of heavy and light chains.

9. CONSTRUCTION OF CHIMERIC GENES

Construction of chimeric genes followed a previously described strategy[Whittle et al (ref. 13)]. A restriction site near the 3′ end of thevariable domain sequence is identified and used to attach anoligonucleotide adapter coding for the remainder of the mouse variableregion and a suitable restriction site for attachment to the constantregion of choice.

9.1. LIGHT CHAIN GENE CONSTRUCTION

The mouse light chain cDNA sequence contains an Aval site near the 3′end of the variable region [FIG. 1(a)]. The majority of the sequence ofthe variable region was isolated as a 396 bp. EcoR1-Aval fragment. Anoligonucleotide adapter was designed to replace the remainder of the 3′region of the variable region from the Aval site and to include the 5′residues of the human constant region up to and including a unique Nar1site which had been previously engineered into the constant region.

A Hind111 site was introduced to act as a marker for insertion of thelinker.

The linker was ligated to the V_(L) fragment and the 413 bp EcoR1-Nar1adapted fragment was purified from the ligation mixture.

The constant region was isolated as an Nar1-BamH1 fragment from an M13clone NW361 and was ligated with the variable region DNA into anEcoR1/BamH1/C1P pSP65 treated vector in a three way reaction to yieldplasmid JA143. Clones were isolated after transformation into E. coliand the linker and junction sequences were confirmed by the presence ofthe Hind111 site and by DNA sequencing.

9.2 LIGHT CHAIN GENE CONSTRUCTION—VERSION 2

The construction of the first chimeric light chain gene produces afusion of mouse and human amino acid sequences at the variable-constantregion junction. In the case of the OKT3 light chain the amino acids atthe chimera junction are:

This arrangement of sequence introduces a potential site for Asparagine(Asn) linked (N-linked) glycosylation at the V-C junction. Therefore, asecond version of the chimeric light chain oligonucleotide adapter wasdesigned in which the threonine (Thr), the first amino acid of the humanconstant region, was replaced with the equivalent amino acid from themouse constant region, Alanine (Ala).

An internal Hind111 site was not included in this adapter, todifferentiate the two chimeric light chain genes.

The variable region fragment was isolated as a 376 bp EcoR1-Avalfragment. The oligonucleotide linker was ligated to Nar1 cut pNW361 andthen the adapted 396 bp constant region was isolated after recutting themodified pNW361 with EcoR1. The variable region fragment and themodified constant region fragment were ligated directly into EcoR1/C1Ptreated pEE6hCMVneo to yield pJA137.

Initially all clones examined had the insert in the incorrectorientation. Therefore, the insert was re-isolated and recloned to turnthe insert round and yield plasmid pJA141. Several clones with theinsert in the correct orientation were obtained and the adapter sequenceof one was confirmed by DNA sequencing

9.3. HEAVY CHAIN GENE CONSTRUCTION

9.3.1. CHOICE OF HEAVY CHAIN GENE ISOTYPE

The constant region isotype chosen for the heavy chain was human IgG4.

9.3.2. GENE CONSTRUCTION

The heavy chain cDNA sequence showed a Ban1 site near the 3′ end of thevariable region [FIG. 2(a)].

The majority of the sequence of the variable region was isolated as a426 bp. EcoR1/C1P/Ban1 fragment. An oligonucleotide adapter wasdesignated to replace the remainder of the 3′ region of the variableregion from the Ban1 site up to and including a unique HindIII sitewhich had been previously engineered into the first two amino acids ofthe constant region.

The linker was ligated to the V_(H) fragment and the EcoR1-Hind111adapted fragment was purified from the ligation mixture.

The variable region was ligated to the constant region by cutting pJA91(ref. ??) with EcoR1 and Hind111 removing the intron fragment andreplacing it with the V_(H) to yield pJA142. Clones were isolated aftertransformation into E. coli JM101 and the linker and junction sequenceswere confirmed by DNA sequencing. (N.B. The Hind111 site is lost oncloning).

10. CONSTRUCTION OF CHIMERIC EXPRESSION VECTORS

10.1. neo AND gpt VECTORS

The chimeric light chain (version 1) was removed from pJA143 as an EcoR1fragment and cloned into EcoR1/C1P treated pEE6hCMVneo expression vectorto yield pJA145. Clones with the insert in the correct orientation wasidentified by restriction mapping.

The chimeric light chain (version 2) was constructed as described above.

The chimeric heavy chain gene was isolated from pJA142 as a 2.5 KbpEcoR1/BamH1 fragment and cloned into the EcoR1/Bc11/C1P treated vectorfragment of a derivative of pEE6hCMVgpt to yield plasmid pJA144.

10.2. GS SEPARATE VECTORS

GS versions of pJA141 and pJA144 were constructed by replacing the neoand gpt cassettes by a BamH1/Sa11/C1P treatment of the plasmids,isolation of the vector fragment and ligation to a GS-containingfragment from the plasmid pRO49 to yield the light chain vector pJA179and the heavy chain vector pJA180.

10.3. GS SINGLE VECTOR CONSTRUCTION

Single vector constructions containing the cL (chimeric light), cH(chimeric heavy) and GS genes on one plasmid in the order cL-cH-GS, orcH-cL-GS and with transcription of the genes being head to tail e.g.cL>cH>GS were constructed. These plasmids were made by treating pJA179or pJA180 with BamH1/C1P and ligating in a Bg111/Hind111 hCMV promotercassette along with either the Hind111/ BamH1 fragment from pJA141 intopJA180 to give the cH-cL-GS plasmid pJA182 or the Hind111/BamH1 fragmentfrom pJA144 into pJA179 to give the cL-CH-GS plasmid pJA181.

11. EXPRESSION OF CHIMERIC GENES

11.1. EXPRESSION IN COS CELLS

The chimeric antibody plasmid pJA145 (cL) and pJA144 (cH) wereco-transfected in COS cells and supernatant from the transientexpression experiment was shown to contain assembled antibody whichbound to the HUT 78 human T-cell line. Metabolic labelling experimentsusing ³⁵S methionine showed expression and assembly of heavy and lightchains. However the light chain mobility seen on reduced gels suggestedthat the potential glycosylation site was being glycosylated. Expressionin COS cells in the presence of tunicamycin showed a reduction in sizeof the light chain to that shown for control chimeric antibodies and theOKT3 mouse light chain. Therefore JA141 was constructed and expressed.In this case the light chain did not show an aberrant mobility or a sizeshift in the presence or absence of tunicamycin. This second version ofthe chimeric light chain, when expressed in association with chimericheavy (cH) chain, produced antibody which showed good binding to HUT 78cells. In both cases antigen binding was equivalent to that of the mouseantibody.

11.2 EXPRESSION IN CHINESE HAMSTER OVARY (CHO) CELLS

Stable cell lines have been prepared from plasmids PJA141/pJA144 andfrom pJA179/pJA180, pJA181 and pJA182 by transfection into CHO cells.

12. CDR-GRAFTING

The approach taken was to try to introduce sufficient mouse residuesinto a human variable region framework to generate antigen bindingactivity comparable to the mouse and chimeric antibodies.

12.1. VARIABLE REGION ANALYSIS

From an examination of a small database of structures of antibodies andantigen-antibody complexes it is clear that only a small number ofantibody residues make direct contact with antigen. Other residues maycontribute to antigen binding by positioning the contact residues infavourable configurations and also by inducing a stable packing of theindividual variable domains and stable interaction of the light andheavy chain variable domains. The residues chosen for transfer can beidentified in a number of ways:

(a) By examination of antibody X-ray crystal structures the antigenbinding surface can be predominantly located on a series of loops, threeper domain, which extend from the B-barrel framework.

(b) By analysis of antibody variable domain sequences regions ofhypervariability [termed the Complementarity Determining Regions (CDRs)by Wu and Kabat (ref. 5)] can be identified. In the most but not allcases these CDRs correspond to, but extend a short way beyond, the loopregions noted above.

(c) Residues not identified by (a) and (b) may contribute to antigenbinding directly or indirectly by affecting antigen binding sitetopology, or by inducing a stable packing of the individual variabledomains and stabilising the inter-variable domain interaction. Theseresidues may be identified either by superimposing the sequences for agiven antibody on a known structure and looking at key residues fortheir contribution, or by sequence alignment analysis and noting“idiosyncratic” residues followed by examination of their structurallocation and likely effects.

12.1.1. LIGHT CHAIN

FIG. 3 shows an alignment of sequences for the human framework regionRE1 and the OKT3 light variable region. The structural loops (LOOP) andCDRs (KABAT) believed to correspond to the antigen binding region aremarked. Also marked are a number of other residues which may alsocontribute to antigen binding as described in 13.1(c). Above thesequence in FIG. 3 the residue type indicates the spatial location ofeach residue side chain, derived by examination of resolved structuresfrom X-ray crystallography analysis. The key to this residue typedesignation is as follows:

N—near to CDR (From X-ray Structures)

P—Packing

S—Surface

I—Interface

—Packing/Part Exposed

B—Buried Non-Packing

E—Exposed

*—Interface

?—Non-CDR Residues which may require to be left as Mouse sequence.

Residues underlined in FIG. 3 are amino acids. RE1 was chosen as thehuman framework because the light chain is a kappa chain and the kappavariable regions show higher homology with the mouse sequences than alambda light variable region, e.g. KOL (see below). RE1 was chosen inpreference to another kappa light chain because the X-ray structure ofthe light chain has been determined so that a structural examination ofindividual residues could be made.

12.1.2. HEAVY CHAIN

Similarly FIG. 4 shows an alignment of sequences for the human frameworkregion KOL and the OKT3 heavy variable region. The structural loops andCDRs believed to correspond to the antigen binding region are marked.Also marked are a number of other residues which may also contribute toantigen binding as described in 12.1(c). The residue type key and otherindicators used in FIG. 4 are the same as those used in FIG. 3.

KOL was chosen as the heavy chain framework because the X-ray structurehas been determined to a better resolution than, for example, NEWM andalso the sequence alignment of OKT3 heavy variable region showed aslightly better homology to KOL than to NEWM.

12.2. DESIGN OF VARIABLE GENES

The variable region domains were designed with mouse variable regionoptimal codon usage [Grantham and Perrin (ref. 15)] and used the B72.3signal sequences [Whittle et al (ref. 13)]. The sequences were designedto be attached to the constant region in the same way as for thechimeric genes described above. Some constructs contained the “Kozakconsensus sequence” [Kozak (ref. 16)] directly linked to the 5′ of thesignal sequence in the gene. This sequence motif is believed to have abeneficial role in translation initiation in eukaryotes.

12.3. GENE CONSTRUCTION

To build the variable regions, various strategies are available. Thesequence may be assembled by using oligonucleotides in a manner similarto Jones et al (ref. 17) or by simultaneously replacing all of the CDRsor loop regions by oligonucleotide directed site specific mutagenesis ina manner similar to Verhoeyen et al (ref. 2). Both strategies were usedand a list of constructions is set out in Tables 1 and 2 and FIGS. 4 and5. It was noted in several cases that the mutagenesis approach led todeletions and rearrangements in the gene being remodelled, while thesuccess of the assembly approach was very sensitive to the quality ofthe oligonucleotides.

13. CONSTRUCTION OF EXPRESSION VECTORS

Genes were isolated from M13 or SP65 based intermediate vectors andcloned into pEE6hCMVneo for the light chains and pEE6hCMVgpt for theheavy chains in a manner similar to that for the chimeric genes asdescribed above.

TABLE 1 CDR-GRAFTED GENE CONSTRUCTS KOZAK MOUSE SEQUENCE METHOD OFSEQUENCE CODE CONTENT CONSTRUCTION − + LIGHT CHAIN  ALL HUMAN FRAMEWORKRE1 121 26-32, 50-56, 91-96 inclusive SDM and gene assembly + n.d. 121A26-32, 50-56, 91-96 inclusive Partial gene assembly n.d. + +1, 3, 46, 47121B 26-32, 50-56, 91-96 inclusive Partial gene assembly n.d. + +46, 47221 24-24, 50-56, 91-96 inclusive Partial gene assembly + + 221A 24-34,50-56, 91-96 inclusive Partial gene assembly + + +1, 3, 46, 47 221B24-34, 50-56, 91-96 inclusive Partial gene assembly + + +1, 3 221C24-34, 50-56, 91-96 inclusive Partial gene assembly + + HEAVY CHAIN  ALLHUMAN FRAMEWORK KOL 121 26-32, 50-56, 95-100B inclusive Gene assemblyn.d. + 131 26-32, 50-58, 95-100B inclusive Cene assembly n.d. + 14126-32, 50-65, 95-100B inclusive Partial gene assembly + n.d. 321 26-35,50-56, 95-100B inclusive Partial gene assembly + n.d. 331 26-35, 50-58,95-100B inclusive Partial gene assembly + Gene assembly + 341 26-35,50-65, 95-100B inclusive SDM + Partial gene assembly + 341A 26-35,50-65, 95-100B inclusive Gene assembly n.d. + +6, 23, 24, 48, 49, 71,73, 76, 78, 88, 91 (+63 = human) 341B 26-35, 50-65, 95-100B inclusiveGene assembly n.d. + +48, 49, 71, 73, 76, 78, 88, 91 (+63 + human) KEYn.d. not done SDM Site directed mutagenesis Gene assembly Variableregion assembled entirely from oligonucleotides Partial geneassembly Variable region assembled by combination of restrictionfragments either from other genes originally created by SDM and geneassembly or by oligonucleotide assembly of part of the variable regionand reconstruction with restriction fragments from other genesoriginally created by SDM and gene assembly

14. EXPRESSION OF CDR-GRAFTED GENES

14.1. PRODUCTION OF ANTIBODY CONSISTING OF GRAFTED LIGHT (gL) CHAINSWITH MOUSE HEAVY (mH) OR CHIMERIC HEAVY (cH) CHAINS

All gL chains, in association with mH or cH produced reasonable amountsof antibody. Insertion of the Kozak consensus sequence at a position 5′to the ATG (kgL constructs) however, led to a 2-5 fold improvement innet expression. Over an extended series of experiments expression levelswere raised from approximately 200 ng/ml to approximately 500 ng/ml forkgL/cH or kgL/mH combinations.

When direct binding to antigen on HUT 78 cells was measured, a constructdesigned to include mouse sequence based on loop length (gL121) did notlead to active antibody in association with mH or cH. A constructdesigned to include mouse sequence based on Kabat CDRs (gL221)demonstrated some weak binding in association with mH or cH. However,when framework residues 1, 3, 46, 47 were changed from the human to themurine OKT3 equivalents based on the arguments outlined in Section 12.1antigen binding was demonstrated when both of the new constructs, whichwere termed 121A and 221A were co-expressed with cH. When the effects ofthese residues were examined in more detail, it appears that residues 1and 3 are not major contributing residues as the product of the gL221Bgene shows little detectable binding activity in association with cH.The light chain product of gL221C, in which mouse sequences are presentat 46 and 47, shows good binding activity in association with cH.

14.2 PRODUCTION OF ANTIBODY CONSISTING OF GRAFTED HEAVY (gH) CHAINS WITHMOUSE LIGHT (mL) OR CHIMERIC LIGHT (cL) CHAINS

Expression of the gH genes proved to be more difficult to achieve thanfor gL. First, inclusion of the Kozak sequence appeared to have nomarked effect on expression of gH genes. Expression appears to beslightly improved but not to the same degree as seen for the graftedlight chain.

Also, it proved difficult to demonstrate production of expectedquantities of material when the loop choice (amino acid 26-32) for CDR1is used, e.g. gH121, 131, 141 and no conclusions can be drawn aboutthese constructs.

Moreover, co-expression of the gH341 gene with cL or mL has beenvariable and has tended to produce lower amounts of antibody than thecH/cL or mH/mL combinations. The alterations to gH341 to produce gH341Aand gH341B lead to improved levels of expression.

This may be due either to a general increase in the fraction of mousesequence in the variable region, or to the alteration at position 63where the residue is returned to the human amino acid Valine (Val) fromPhenylalanine (Phe) to avoid possible internal packing problems with therest of the human framework. This arrangement also occurs in gH331 andgH321.

When gH321 or gH331 were expressed in association with cL, antibody wasproduced but antibody binding activity was not detected.

When the more conservative gH341 gene was used antigen binding could bedetected in association with cL or mL, but the activity was onlymarginally above the background level.

When further mouse residues were substituted based on the arguments in12.1, antigen binding could be clearly demonstrated for the antibodyproduced when kgH341A and kgH341B were expressed in association with cL.

14.3 PRODUCTION OF FULLY CDR-GRAFTED ANTIBODY

The kgL221A gene was co-expressed with kgH341, kgH341A or kgH341B. Forthe combination kgH221A/kgH341 very little material was produced in anormal COS cell expression.

For the combinations kgL221A/kgH341A or kgH221A/kgH341B amounts ofantibody similar to gL/cH was produced.

In several experiments no antigen binding activity could be detectedwith kgH221A/gH341 or kgH221A/kgH341 combinations, although expressionlevels were very low.

Antigen binding was detected when kgL221A/kgH341A or kgH221A/kgH341Bcombinations were expressed. In the case of the antibody produced fromthe kgL221A/kgH341A combination the antigen binding was very similar tothat of the chimeric antibody.

An analysis of the above results is given below.

15. DISCUSSION OF CDR-GRAFTING RESULTS

In the design of the fully humanised antibody the aim was to transferthe minimum number of mouse amino acids that would confer antigenbinding onto a human antibody framework.

15.1. LIGHT CHAIN

15.1.1. EXTENT OF THE CDRs

For the light chain the regions defining the loops known from structuralstudies of other antibodies to contain the antigen contacting residues,and those hypervariable sequences defined by Kabat et al (refs. 4 and 5)as Complementarity Determining Regions (CDRs) are equivalent for CDR2.For CDR1 the hypervariable region extends from residues 24-34 inclusivewhile the structural loop extends from 26-32 inclusive. In the case ofOKT3 there is only one amino acid difference between the two options, atamino acid 24, where the mouse sequence is a serine and the humanframework RE1 has glutamine. For CDR3 the loop extends from residues91-96 inclusive while the Kabat hypervariability extends from residues89-97 inclusive. For OKT3 amino acids 89, 90 and 97 are the same betweenOKT3 and RE1 (FIG. 3). When constructs based on the loop choice for CDR1(gL121) and the Kabat choice (gL221) were made and co-expressed with mHor cH no evidence for antigen binding activity could be found for gL121,but trace activity could be detected for the gL221, suggesting that asingle extra mouse residue in the grafted variable region could havesome detectable effect. Both gene constructs were reasonable wellexpressed in the transient expression system.

15.1.2. FRAMEWORK RESIDUES

The remaining framework residues were then further examined, inparticular amino acids known from X-ray analysis of other antibodies tobe close to the CDRs and also those amino acids which in OKT3 showeddifferences from the consensus framework for the mouse subgroup(subgroup VI) to which OKT3 shows most homology. Four positions 1, 3, 46and 47 were identified and their possible contribution was examined bysubstituting the mouse amino acid for the human amino acid at eachposition.

Therefore gL221A (gL221+D1Q, Q3V, L46R, L47W, see FIG. 3 and Table 1)was made, cloned in EE6hCMVneo and co-expressed with cH (pJA144). Theresultant antibody was well expressed and showed good binding activity.When the related genes gL221B (gL221+D1Q, Q3V) and gL221C (gL221+L46R,L47W) were made and similarly tested, while both genes produced antibodywhen co-expressed with cH, only the gl221C/cH combination showed goodantigen binding. When the gL121A (gL121+D1Q, Q3V, L46R, L47W) gene wasmade and co-expressed with cH, antibody was produced which also bound toantigen.

15.2. HEAVY CHAIN

15.2.1. EXTENT OF THE CDRs

For the heavy chain the loop and hypervariability analyses agree only inCDR3. For CDR1 the loop region extends from residues 26-32 inclusivewhereas the Kabat CDR extends from residues 31-35 inclusive. For CDR2the loop region is from 50-58 inclusive while the hypervariable regioncovers amino acids 50-65 inclusive. Therefore humanised heavy chainswere constructed using the framework from antibody KOL and with variouscombinations of these CDR choices, including a shorter choice for CDR2of 50-56 inclusive as there was some uncertainty as to the definition ofthe end point for the CDR2 loop around residues 56 to 58. The genes wereco-expressed with mL or cL initially. In the case of the gH genes withloop choices for CDR1 e.g. gH121, gH131, gH141 very little antibody wasproduced in the culture supernatants. As no free light chain wasdetected it was presumed that the antibody was being made and assembledinside the cell but that the heavy chain was aberrant in some way,possibly incorrectly folded, and therefore the antibody was beingdegraded internally. In some experiments trace amounts of antibody couldbe detected in ³⁵S labelling studies.

As no net antibody was produced, analysis of these constructs was notpursued further. When, however, a combination of the loop choice and theKabat choice for CDR1 was tested (mouse amino acids 26-35 inclusive) andin which residues 31 (Ser to Arg), 33 (Ala to Thr), and 35 (Tyr to His)were changed from the human residues to the mouse residue and comparedto the first series, antibody was produced for gH321, kgH331 and kgH341when co-expressed with cL.

Expression was generally low and could not be markedly improved by theinsertion of the Kozak consensus sequence 5′ to the ATG of the signalsequence of the gene, as distinct from the case of the gL genes wheresuch insertion led to a 2-5 fold increase in net antibody production.However, only in the case of gH341/mL or kgH341/cL could marginalantigen binding activity be demonstrated. When the kgH341 gene wasco-expressed with kgL221A, the net yield of antibody was too low to givea signal above the background level in the antigen binding assay.

15.2.2. FRAMEWORK RESIDUES

As in the case of the light chain the heavy chain frameworks werere-examined. Possibly because of the lower initial homology between themouse and human heavy variable domains compared to the light chains,more amino acid positions proved to be of interest. Two genes kgH341Aand kgH341B were constructed, with 11 or 8 human residues respectivelysubstituted by mouse residues compared to gH341, and with the CDR2residue 63 returned to the human amino acid potentially to improvedomain packing. Both showed antigen binding when combined with cL orkgL221A, the kgH341A gene with all 11 changes appearing to be thesuperior choice.

15.3 INTERIM CONCLUSIONS

It has been demonstrated, therefore, for OKT3 that the transfer antigenbinding ability to the humanised antibody, mouse residues outside theCDR regions defined by the Kabat hypervariability or structural loopchoices are required for both the light and heavy chains. Fewer extraresidues are needed for the light chain, possibly due to the higherinitial homology between the mouse and human kappa variable regions. Ofthe changes seven (1 and 3 from the light chain and 6, 23, 71, 73 and 76from the heavy chain) are predicted from a knowledge of other antibodystructures to be either partly exposed or on the antibody surface. Ithas been shown here that residues 1 and 3 in the light chain are notabsolutely required to be the mouse sequence; and for the heavy chainthe gH341B heavy chain in combination with the 221A light chaingenerated only weak binding activity. Therefore the presence of the 6and 23 and 24 changes are important to maintain binding affinity similarto that of murine antibody. It was important, therefore, to furtherstudy the individual contribution of the other 8 mouse residues of thekgH341A gene compared to kgH341.

16. FURTHER CDR-GRAFTING EXPERIMENTS

Additional CDR-grafted heavy chain genes were prepared substantially asdescribed above. With reference to Table 2 the further heavy chain geneswere based upon the gh341 (plasmid pJA178) and gH341A (plasmid pJA185)with either mouse OKT3 or human KOL residues at 6, 23, 24, 48, 49, 63,71, 73, 76, 78, 88 and 91, as indicated. The CDR-grafted light chaingenes used in these further experiments were gL221, gL221A, gL221B andgL221C as described above.

TABLE 2 OKT3 HEAVY CHAIN CDR GRAFTS 1. gH341 and derivatives RES NUM6 23 24 48 49 63 71 73 76 78 88 91 OKT3vhQ  K  A  I  G  F  T  K  S  A  A  Y gH341E  S  S  V  A  F  R  N  N  L  G  F JA178 gH341AQ  K  A  I  G  V  T  K  S  A  A  Y JA185 gH341EQ  K  A  I  G  V  T  K  S  A  G  G JA198 gH341*Q  K  A  I  G  V  T  K  N  A  G  F JA207 gH341*Q  K  A  I  G  V  R  N  N  A  G  F JA209 gH341DQ  K  A  I  G  V  T  K  N  L  G  F JA197 gH341*Q  K  A  I  G  V  R  N  N  L  G  F JA199 gH341CQ  K  A  V  A  F  R  N  N  L  G  F JA184 gH341*Q  S  A  I  G  V  T  K  S  A  A  Y JA203 gH341*E  S  A  I  G  V  T  K  S  A  A  Y JA205 gH341BE  S  S  I  G  V  T  K  S  A  A  Y JA183 gH341*Q  S  A  I  G  V  T  K  S  A  G  F JA204 gH341*E  S  A  I  G  V  T  K  S  A  G  F JA206 gH341*Q  S  A  I  G  V  T  K  N  A  G  F JA208 KOLE  S  S  V  A     R  N  N  L  G  F OKT3 LIGHT CHAIN CDR GRAFTS 2. gL221and derivatives RES NUM 1 3 46 47 OKT3v1 Q V  R  W GL221 D Q  L  L DA221gL221A Q V  R  W DA221A gL221B Q V  L  L DA221B GL221C D Q  R  W DA221CRE1 D Q  L  L MURINE RESIDUES ARE UNDERLINED

The CDR-grafted heavy and light chain genes were co-expressed in COScells either with one another in various combinations but also with thecorresponding murine and chimeric heavy and light chain genessubstantially as described above. The resultant antibody products werethen assayed in binding and blocking assays with HPB-ALL cells asdescribed above.

The results of the assays for various grafted heavy chains co-expressedwith the gL221C light chain are given in FIGS. 7 and 8 (for the JA184,JA185, JA197 and JA198 constructs—see Table 2), in FIG. 9 (for theJA183, JA184, JA185 and JA197 constructs) in FIG. 10 (for the chimeric,JA185, JA199, JA204, JA205, JA207, JA208 and JA209 constructs) and inFIG. 11 (for the JA183, JA184, JA185, JA198, JA203, JA205 and JA206constructs).

The basic grafted product without any human to murine changes in thevariable frameworks, i.e. gL221 co-expressed with gh341 (JA178), andalso the “fully grafted” product, having most human to murine changes inthe grafted heavy chain framework, i.e. gL221C co-expressed with gh341A(JA185), were assayed for relative binding affinity in a competitionassay against murine OKT3 reference standard, using HPB-ALL cells. Theassay used was as described above in section 3.3. The results obtainedare given in FIG. 12 for the basic grafted product and in FIG. 13 forthe fully grafted product. These results indicate that the basic graftedproduct has relatively poor binding abiliaty as compared with the OKT3murine reference standard; whereas the “fully grafted” product has abinding ability very similar to that of the OKT3 murine referencestandard.

The binding and blocking assay results indicate the following:

The JA198 and JA207 constructs appear to have the best bindingcharacteristics and similar binding abilities, both substantially thesame as the chimeric and fully grafted gH341A products. This indicatesthat positions 88 and 91 and position 76 are not highly critical formaintaining the OKT3 binding ability; whereas at least some of positions6, 23, 24, 48, 49, 71, 73 and 78 are more important.

This is borne out by the finding that the JA209 and JA199, although ofsimilar binding ability to one another, are of lower binding abilitythan the JA198 and JA207 constructs. This indicates the importance ofhaving mouse residues at positions 71, 73 and 78, which are eithercompletely or partially human in the JA199 and JA209 constructsrespectively.

Moreover, on comparing the results obtained for the JA205 and JA183constructs it is seen that there is a decrease in binding going from theJA205 to the JA183 constructs. This indicates the importance ofretaining a mouse residue at position 23, the only position changedbetween JA205 and JA183.

These and other results lead us to the conclusion that of the 11 mouseframework residues used in the gH341A (JA185) construct, it is importantto retain mouse residues at all of positions 6, 23, 24, 48 and 49, andpossibly for maximum affinity at positions 71, 73 and 78.

REFERENCES

1. Kohler & Milstein, Nature, 265, 295-497, 1975.

2. Verhoeyen et al, Science, 239, 1534-1536, 1988.

3. Riechmann et al, Nature, 332, 323-324, 1988.

4. Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M., Gottesman, K.S., 1987, in Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, NIH, USA.

5. Wu, T. T., and Kabat, E. A., 1970, J. Exp. Med. 132 211-250.

6. Queen et al, (1989), Proc. Natl. Acad. Sci. USA, 86, 10029-10033 andWO 90/07861

7. Chatenoud et al, (1986), J. Immunol. 137, 830-838.

8. Jeffers et al, (1986), Transplantation, 41, 572-578.

9. Maniatis et al, Molecular Cloning, Cold Spring Harbor, N.Y., 1982.

10. Primrose and Old, Principles of Gene Manipulation, Blackwell,Oxford, 1980.

11. Sanger, F., Nicklen, S., Coulson, A. R., 1977, Proc. Natl. Acad.Sci. USA, 74 5463

12. Kramer, W., Drutsa, V., Jansen, H. -W., Kramer, B., Plugfelder, M.,Fritz, H. -J., 1984, Nucl. Acids Res. 12, 9441

13. Whittle, N., Adair, J., Lloyd, J. C., Jenkins, E., Devine, J.,Schlom, J., Raubitshek, A., Colcher, D., Bodmer, M., 1987, ProteinEngineering 1, 499.

14. Bebbington, C. R., Published International Patent Application WO89/01036.

15. Granthan and Perrin 1986, Immunology Today 7, 160.

16. Kozak, M., 1987, J. Mol. Biol. 196, 947.

17. Jones, T. P., Dear, P. H., Foote, J., Neuberger, M. S., Winter, G.,1986, Nature, 321, 522

29 1 20 DNA Artificial Sequence Description of Artificial Sequence probe1 tccagatgtt aactgctcac 20 2 23 DNA Artificial Sequence Description ofArtificial Sequence probe 2 caggggccag tggatggata gac 23 3 9 PRTArtificial Sequence Description of Artificial Sequence Novel Sequence 3Leu Glu Ile Asn Arg Thr Val Ala Ala 1 5 4 943 DNA Mouse CDS (18)..(722)4 gaattcccaa agacaaa atg gat ttt caa gtg cag att ttc agc ttc ctg 50 MetAsp Phe Gln Val Gln Ile Phe Ser Phe Leu 1 5 10 cta atc agt gcc tca gtcata ata tcc aga gga caa att gtt ctc acc 98 Leu Ile Ser Ala Ser Val IleIle Ser Arg Gly Gln Ile Val Leu Thr 15 20 25 cag tct cca gca atc atg tctgca tct cca ggg gag aag gtc acc atg 146 Gln Ser Pro Ala Ile Met Ser AlaSer Pro Gly Glu Lys Val Thr Met 30 35 40 acc tgc agt gcc agc tca agt gtaagt tac atg aac tgg tac cag cag 194 Thr Cys Ser Ala Ser Ser Ser Val SerTyr Met Asn Trp Tyr Gln Gln 45 50 55 aag tca ggc acc tcc ccc aaa aga tggatt tat gac aca tcc aaa ctg 242 Lys Ser Gly Thr Ser Pro Lys Arg Trp IleTyr Asp Thr Ser Lys Leu 60 65 70 75 gct tct gga gtc cct gct cac ttc aggggc agt ggg tct ggg acc tct 290 Ala Ser Gly Val Pro Ala His Phe Arg GlySer Gly Ser Gly Thr Ser 80 85 90 tac tct ctc aca atc agc ggc atg gag gctgaa gat gct gcc act tat 338 Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala GluAsp Ala Ala Thr Tyr 95 100 105 tac tgc cag cag tgg agt agt aac cca ttcacg ttc ggc tcg ggg aca 386 Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe ThrPhe Gly Ser Gly Thr 110 115 120 aag ttg gaa ata aac cgg gct gat act gcacca act gta tcc atc ttc 434 Lys Leu Glu Ile Asn Arg Ala Asp Thr Ala ProThr Val Ser Ile Phe 125 130 135 cca cca tcc agt gag cag tta aca tct ggaggt gcc tca gtc gtg tgc 482 Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly GlyAla Ser Val Val Cys 140 145 150 155 ttc ttg aac aac ttc tac ccc aaa gacatc aat gtc aag tgg aag att 530 Phe Leu Asn Asn Phe Tyr Pro Lys Asp IleAsn Val Lys Trp Lys Ile 160 165 170 gat ggc agt gaa cga caa aat ggc gtcctg aac agt tgg act gat cag 578 Asp Gly Ser Glu Arg Gln Asn Gly Val LeuAsn Ser Trp Thr Asp Gln 175 180 185 gac agc aaa gac agc acc tac agc atgagc agc acc ctc acg ttg acc 626 Asp Ser Lys Asp Ser Thr Tyr Ser Met SerSer Thr Leu Thr Leu Thr 190 195 200 aag gac gag tat gaa cga cat aac agctat acc tgt gag gcc act cac 674 Lys Asp Glu Tyr Glu Arg His Asn Ser TyrThr Cys Glu Ala Thr His 205 210 215 aag aca tca act tca ccc att gtc aagagc ttc aac agg aat gag tgt 722 Lys Thr Ser Thr Ser Pro Ile Val Lys SerPhe Asn Arg Asn Glu Cys 220 225 230 235 tagagacaaa ggtcctgaga cgccaccaccagctcccagc tccatcctat cttcccttct 782 aaggtcttgg aggcttcccc acaagcgcttaccactgttg cggtgctcta aacctcctcc 842 cacctccttc tcctcctcct ccctttccttggcttttatc atgctaatat ttgcagaaaa 902 tattcaataa agtgagtctt tgccttgaaaaaaaaaaaaa a 943 5 235 PRT Mouse 5 Met Asp Phe Gln Val Gln Ile Phe SerPhe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Ile Ser Arg Gly Gln IleVal Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu LysVal Thr Met Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met Asn TrpTyr Gln Gln Lys Ser Gly Thr Ser 50 55 60 Pro Lys Arg Trp Ile Tyr Asp ThrSer Lys Leu Ala Ser Gly Val Pro 65 70 75 80 Ala His Phe Arg Gly Ser GlySer Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Gly Met Glu Ala Glu AspAla Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Phe ThrPhe Gly Ser Gly Thr Lys Leu Glu Ile Asn 115 120 125 Arg Ala Asp Thr AlaPro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 130 135 140 Gln Leu Thr SerGly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 145 150 155 160 Tyr ProLys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 165 170 175 GlnAsn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 180 185 190Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 195 200205 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 210215 220 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 6 1570DNA Mouse CDS (41)..(1444) 6 gaattcccct ctccacagac actgaaaact ctgactcaacatg gaa agg cac tgg 55 Met Glu Arg His Trp 1 5 atc ttt cta ctc ctg ttgtca gta act gca ggt gtc cac tcc cag gtc 103 Ile Phe Leu Leu Leu Leu SerVal Thr Ala Gly Val His Ser Gln Val 10 15 20 cag ctg cag cag tct ggg gctgaa ctg gca aga cct ggg gcc tca gtg 151 Gln Leu Gln Gln Ser Gly Ala GluLeu Ala Arg Pro Gly Ala Ser Val 25 30 35 aag atg tcc tgc aag gct tct ggctac acc ttt act agg tac acg atg 199 Lys Met Ser Cys Lys Ala Ser Gly TyrThr Phe Thr Arg Tyr Thr Met 40 45 50 cac tgg gta aaa cag agg cct gga cagggt ctg gaa tgg att gga tac 247 His Trp Val Lys Gln Arg Pro Gly Gln GlyLeu Glu Trp Ile Gly Tyr 55 60 65 att aat cct agc cgt ggt tat act aat tacaat cag aag ttc aag gac 295 Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr AsnGln Lys Phe Lys Asp 70 75 80 85 aag gcc aca ttg act aca gac aaa tcc tccagc aca gcc tac atg caa 343 Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser SerThr Ala Tyr Met Gln 90 95 100 ctg agc agc ctg aca tct gag gac tct gcagtc tat tac tgt gca aga 391 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala ValTyr Tyr Cys Ala Arg 105 110 115 tat tat gat gat cat tac tgc ctt gac tactgg ggc caa ggc acc act 439 Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly Thr Thr 120 125 130 ctc aca gtc tcc tca gcc aaa aca aca gcccca tcg gtc tat cca ctg 487 Leu Thr Val Ser Ser Ala Lys Thr Thr Ala ProSer Val Tyr Pro Leu 135 140 145 gcc cct gtg tgt gga gat aca act ggc tcctcg gtg act cta gga tgc 535 Ala Pro Val Cys Gly Asp Thr Thr Gly Ser SerVal Thr Leu Gly Cys 150 155 160 165 ctg gtc aag ggt tat ttc cct gag ccagtg acc ttg acc tgg aac tct 583 Leu Val Lys Gly Tyr Phe Pro Glu Pro ValThr Leu Thr Trp Asn Ser 170 175 180 gga tcc ctg tcc agt ggt gtg cac accttc cca gct gtc ctg cag tct 631 Gly Ser Leu Ser Ser Gly Val His Thr PhePro Ala Val Leu Gln Ser 185 190 195 gac ctc tac acc ctc agc agc tca gtgact gta acc tcg agc acc tgg 679 Asp Leu Tyr Thr Leu Ser Ser Ser Val ThrVal Thr Ser Ser Thr Trp 200 205 210 ccc agc cag tcc atc acc tgc aat gtggcc cac ccg gca agc agc acc 727 Pro Ser Gln Ser Ile Thr Cys Asn Val AlaHis Pro Ala Ser Ser Thr 215 220 225 aag gtg gac aag aaa att gag ccc agaggg ccc aca atc aag ccc tgt 775 Lys Val Asp Lys Lys Ile Glu Pro Arg GlyPro Thr Ile Lys Pro Cys 230 235 240 245 cct cca tgc aaa tgc cca gca cctaac ctc ttg ggt gga cca tcc gtc 823 Pro Pro Cys Lys Cys Pro Ala Pro AsnLeu Leu Gly Gly Pro Ser Val 250 255 260 ttc atc ttc cct cca aag atc aaggat gta ctc atg atc tcc ctg agc 871 Phe Ile Phe Pro Pro Lys Ile Lys AspVal Leu Met Ile Ser Leu Ser 265 270 275 ccc ata gtc aca tgt gtg gtg gtggat gtg agc gag gat gac cca gat 919 Pro Ile Val Thr Cys Val Val Val AspVal Ser Glu Asp Asp Pro Asp 280 285 290 gtc cag atc agc tgg ttt gtg aacaac gtg gaa gta cac aca gct cag 967 Val Gln Ile Ser Trp Phe Val Asn AsnVal Glu Val His Thr Ala Gln 295 300 305 aca caa acc cat aga gag gat tacaac agt act ctc cgg gtg gtc agt 1015 Thr Gln Thr His Arg Glu Asp Tyr AsnSer Thr Leu Arg Val Val Ser 310 315 320 325 gcc ctc ccc atc cag cac caggac tgg atg agt ggc aag gag ttc aaa 1063 Ala Leu Pro Ile Gln His Gln AspTrp Met Ser Gly Lys Glu Phe Lys 330 335 340 tgc aag gtc aac aac aaa gacctc cca gcg ccc atc gag aga acc atc 1111 Cys Lys Val Asn Asn Lys Asp LeuPro Ala Pro Ile Glu Arg Thr Ile 345 350 355 tca aaa ccc aaa ggg tca gtaaga gct cca cag gta tat gtc ttg cct 1159 Ser Lys Pro Lys Gly Ser Val ArgAla Pro Gln Val Tyr Val Leu Pro 360 365 370 cca cca gaa gaa gag atg actaag aaa cag gtc act ctg acc tgc atg 1207 Pro Pro Glu Glu Glu Met Thr LysLys Gln Val Thr Leu Thr Cys Met 375 380 385 gtc aca gac ttc atg cct gaagac att tac gtg gag tgg acc aac aac 1255 Val Thr Asp Phe Met Pro Glu AspIle Tyr Val Glu Trp Thr Asn Asn 390 395 400 405 ggg aaa aca gag cta aactac aag aac act gaa cca gtc ctg gac tct 1303 Gly Lys Thr Glu Leu Asn TyrLys Asn Thr Glu Pro Val Leu Asp Ser 410 415 420 gat ggt tct tac ttc atgtac agc aag ctg aga gtg gaa aag aag aac 1351 Asp Gly Ser Tyr Phe Met TyrSer Lys Leu Arg Val Glu Lys Lys Asn 425 430 435 tgg gtg gaa aga aat agctac tcc tgt tca gtg gtc cac gag ggt ctg 1399 Trp Val Glu Arg Asn Ser TyrSer Cys Ser Val Val His Glu Gly Leu 440 445 450 cac aat cac cac acg actaag agc ttc tcc cgg act ccg ggt aaa 1444 His Asn His His Thr Thr Lys SerPhe Ser Arg Thr Pro Gly Lys 455 460 465 tgagctcagc acccacaaaa ctctcaggtccaaagagaca cccacactca tctccatgct 1504 tcccttgtat aaataaagca cccagcaatgcctgggacca tgtaaaaaaa aaaaaaaaag 1564 gaattc 1570 7 468 PRT Mouse 7 MetGlu Arg His Trp Ile Phe Leu Leu Leu Leu Ser Val Thr Ala Gly 1 5 10 15Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg 20 25 30Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn 65 70 7580 Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser 85 9095 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100105 110 Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr AlaPro 130 135 140 Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr GlySer Ser 145 150 155 160 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe ProGlu Pro Val Thr 165 170 175 Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser GlyVal His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Asp Leu Tyr Thr LeuSer Ser Ser Val Thr Val 195 200 205 Thr Ser Ser Thr Trp Pro Ser Gln SerIle Thr Cys Asn Val Ala His 210 215 220 Pro Ala Ser Ser Thr Lys Val AspLys Lys Ile Glu Pro Arg Gly Pro 225 230 235 240 Thr Ile Lys Pro Cys ProPro Cys Lys Cys Pro Ala Pro Asn Leu Leu 245 250 255 Gly Gly Pro Ser ValPhe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 260 265 270 Met Ile Ser LeuSer Pro Ile Val Thr Cys Val Val Val Asp Val Ser 275 280 285 Glu Asp AspPro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 290 295 300 Val HisThr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 305 310 315 320Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 325 330335 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro 340345 350 Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln355 360 365 Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys GlnVal 370 375 380 Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp IleTyr Val 385 390 395 400 Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn TyrLys Asn Thr Glu 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe MetTyr Ser Lys Leu Arg 420 425 430 Val Glu Lys Lys Asn Trp Val Glu Arg AsnSer Tyr Ser Cys Ser Val 435 440 445 Val His Glu Gly Leu His Asn His HisThr Thr Lys Ser Phe Ser Arg 450 455 460 Thr Pro Gly Lys 465 8 107 PRTMouse 8 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser TyrMet 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp IleTyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg GlySer 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu AlaGlu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn ProPhe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg 100 105 9108 PRT Homo sapiens 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser GlnAsp Ile Ile Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Thr Pro Gly Lys AlaPro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Asn Leu Gln Ala Gly Val ProSer Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr IleSer Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln GlnTyr Gln Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Gln IleThr Arg 100 105 10 118 PRT Mouse 10 Gln Val Gln Leu Gln Gln Ser Gly AlaGlu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys AlaSer Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln ArgPro Gln Gly Leu Glu Trp Ile Gly 35 40 45 Tyr Ile Asn Pro Ser Arg Gly TyrThr Asn Thr Asn Gln Lys Phe Lys 50 55 60 Asp Lys Ala Thr Leu Thr Thr AspLys Ser Ser Ser Thr Ala Tyr Met 65 70 75 80 Gln Leu Ser Ser Leu Thr SerGlu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Tyr Tyr Asp Asp His TyrCys Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser115 11 126 PRT Homo sapiens 11 Gln Val Gln Leu Val Glu Ser Gly Gly GlyVal Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ser SerGly Phe Ile Phe Ser Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala ProGly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Trp Asp Asp Gly Ser AspGln His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg AspAsn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg ProGlu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Arg Asp Gly Gly His Gly PheCys Ser Ser Ala Ser Cys Phe Gly 100 105 110 Pro Asp Tyr Trp Gly Gln GlyThr Pro Val Thr Val Ser Ser 115 120 125 12 119 PRT Artificial SequenceDescription of Artificial Sequence humanized antibody 12 Gln Val Gln LeuVal Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu ArgLeu Ser Cys Ser Ser Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met HisTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr IleAsn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp ArgPhe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu GlnMet Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala ArgTyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 ThrThr Leu Thr Val Ser Ser 115 13 119 PRT Artificial Sequence Descriptionof Artificial Sequence humanized antibody 13 Gln Val Gln Leu Val Gln SerGly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser CysLys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro SerArg Gly Tyr Thr Asn Tyr Asn Gln Lys Val 50 55 60 Lys Asp Arg Phe Thr IleSer Thr Asp Lys Ser Lys Ser Thr Ala Phe 65 70 75 80 Leu Gln Met Asp SerLeu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr AspAsp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu ThrVal Ser Ser 115 14 119 PRT Artificial Sequence Description of ArtificialSequence humanized antibody 14 Gln Val Gln Leu Val Gln Ser Gly Gly GlyVal Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala SerGly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala ProGly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly TyrThr Asn Tyr Asn Gln Lys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Thr AspLys Ser Lys Ser Thr Ala Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg ProGlu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His TyrCys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser115 15 119 PRT Artificial Sequence Description of Artificial Sequencehumanized antibody 15 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val ValGln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly TyrThr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly LysGly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr AsnTyr Asn Gln Lys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Thr Asp Lys SerLys Asn Thr Ala Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu AspThr Gly Val Tyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys LeuAsp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 16119 PRT Artificial Sequence Description of Artificial Sequence humanizedantibody 16 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn ThrAla Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly ValTyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 17 119 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 17 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Thr Asp Lys Ser Lys Asn ThrLeu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly ValTyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 18 119 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 18 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn ThrLeu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly ValTyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 19 119 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 19 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val 35 40 45 Ala Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn ThrLeu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly ValTyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Ser Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 20 119 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 20 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Thr Asp Lys Ser Lys Ser ThrAla Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala ValTyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 21 119 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 21 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Thr Asp Lys Ser Lys Ser ThrAla Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala ValTyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 22 119 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 22 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ser Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Thr Asp Lys Ser Lys Ser ThrAla Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala ValTyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 23 119 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 23 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Thr Asp Lys Ser Lys Ser ThrAla Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly ValTyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 24 119 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 24 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Thr Asp Lys Ser Lys Ser ThrAla Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly ValTyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 25 119 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 25 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Tyr Thr Phe ThrArg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn GlnLys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Thr Asp Lys Ser Lys Asn ThrAla Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly ValTyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 26 107 PRTArtificial Sequence Description of Artificial Sequence humanizedantibody 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val SerTyr Met 20 25 30 Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu GlnPro Glu 65 70 75 80 Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser AsnPro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg 100 10527 107 PRT Artificial Sequence Description of Artificial Sequencehumanized antibody 27 Gln Ile Val Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser SerSer Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala ProLys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro SerArg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile SerSer Leu Gln Pro Glu 65 70 75 80 Asp Ile Ala Thr Tyr Tyr Cys Gln Gln TrpSer Ser Asn Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Gln Ile ThrArg 100 105 28 107 PRT Artificial Sequence Description of ArtificialSequence humanized antibody 28 Gln Ile Val Met Thr Gln Ser Pro Ser SerLeu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser AlaSer Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Thr Pro Gly LysAla Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly ValPro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Phe ThrIle Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Ile Ala Thr Tyr Tyr Cys GlnGln Trp Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu GlnIle Thr Arg 100 105 29 107 PRT Artificial Sequence Description ofArtificial Sequence humanized antibody 29 Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile ThrCys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln ThrPro Gly Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu AlaSer Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp TyrThr Phe Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Ile Ala Thr TyrTyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Gln Gly ThrLys Leu Gln Ile Thr Arg 100 105

What is claimed is:
 1. An anti-CD3 antibody molecule comprising: aCDR-grafted heavy chain wherein, according to the Kabat numberingsystem, residues 31 to 35, 50 to 65 and 95 to 102 are donor residues;and a complementary light chain said CDR-grafted heavy chain having avariable domain comprising predominantly human acceptor antibody heavychain framework residues and murine donor antibody heavy chainantigen-binding residues, said murine donor antibody having affinity forCD3, wherein, according to the Kabat numbering system, in saidCDR-grafted heavy chain, amino acid residues 23, 24, and 49 at least areadditionally donor residues.
 2. The antibody molecule of claim 1 whereinamino acid residues 26 to 30 in said CDR-grafted heavy chain areadditionally donor residues.
 3. The antibody molecule of claim 1 orclaim 2 wherein amino acid residues 71, 73 and 78 in said CDR-graftedheavy chain are additionally donor residues.
 4. The antibody molecule ofclaim 1 wherein at least one of amino acid residues 1, 3, and 76 in saidCDR-grafted heavy chain is additionally a donor residue.
 5. The antibodymolecule of claim 1 wherein at least one of amino acid residues 36, ifnot a tryptophan, 94, if not an arginine, 104 and 106, if not glycines,and 107, if not a threonine, in said CDR-grafted heavy chain areadditionally donor residues.
 6. The antibody molecule of claim 5 whereinat least one of amino acid residues 2, 4, 6, 38, 48, 67, and 69 in saidCDR-grafted heavy chain is additionally a donor residue.
 7. The antibodymolecule of claim 1 wherein said complementary light chain is aCDR-grafted light chain wherein, according to the Kabat numberingsystem, residues 24 to 34, 50 to 56 and 89 to 97 are donor residues,said CDR-grafted light chain having a variable domain comprisingpredominantly human acceptor antibody light chain framework residues andmurine donor antibody light chain antigen-binding residues, said murinedonor antibody having affinity for CD3, wherein, according to the Kabatnumbering system, in said CDR-grafted light chain, amino acid residues46, 48, 58 and 71 at least are donor residues.
 8. A therapeutic ordiagnostic composition comprising the antibody molecule of claim 1 incombination with a pharmaceutically acceptable carrier, diluent orexcipient.
 9. An anti-CD3 antibody molecule comprising a CDR-graftedheavy chain wherein, according to the Kabat numbering system, residues26 to 35, 50 to 65 and 95 to 102 are donor residues; and a complementarylight chain said CDR-grafted heavy chain having a variable domaincomprising predominantly human acceptor antibody heavy chain frameworkresidues and murine donor antibody heavy chain antigen-binding residues,said murine donor antibody having affinity for CD3, wherein, accordingto the Kabat numbering system, in said CDR-grafted heavy chain, aminoacid residues 23, 24, and 49 at least are additionally donor residues.